One barrier that all disease-causing organisms must overcome in order to affect their host is that of microbial communities that live on and within the host (the host microbiome). Yet most studies of infectious disease fail to examine associations between pathogens and the microbiome. Here, we propose using Clostridium difficile as a model for examining how an opportunistic pathogen either causes or takes advantage of changes in the intestinal microbiome to infect the host and cause disease. C. difficile causes serious intestinal disease associated with diarrhea and colitis. C. difficile associated disease (CDAD) is linked with antibiotic treatment regimes and is increasing in incidence, with many hospital associated (nosocomial) outbreaks and continued sequelae. Current theory implies that clinical treatment with antibiotics for other infections disturbs the normal intestinal flora, alloing C. difficile to become established as an invader. While there is evidence to support this etiology, the details of how bacterial overgrowth happens, why many patients do not recover fully after treatment and why, in some patients, CDAD recurs, is still unknown. It is important to examine in detail the changes to the intestinal microbiome that contribute to disease caused by opportunistic pathogens such as C. difficile, as well as to elucidate potential interactions between the intestinal microbiome and pathogens. The language used to describe C. difficile overgrowth in the intestine is similar to that used by invasion ecologists to describe invasion events in large ecosystems. By considering the intestinal microbiome as an ecosystem and combining techniques commonly used in microbial ecology with sampling and analysis techniques from invasion and landscape ecology, a better understanding of how opportunistic pathogens invade and cause disease can be achieved. Use of a recently developed mouse model combined with deep sequencing analysis of different intestinal compartments will determine if C. difficile is distributed non- randomly with regard to location and bacterial taxa i the GI tract, and whether these bacterial associations affect where C. difficile is active which of its various disease states is manifested. In addition, fluorescence in situ hybridization (FISH) wil be used to perform a detailed analysis of the spatial organization of C. difficile with respect to specific microbiome taxa during disease progression and treatment. Aim 1: Comparison using deep sequencing and metagenomic analysis of bacterial communities in five different areas of the intestine, during antibiotic therapy, during the different states of CDAD and in normal controls. Aim 2: Spatial mapping of bacterial populations within the intestine using FISH with a special emphasis on individual species that have positive and negative associations with C. difficile. Aim 3: Advancing the NIAID AREA mission will be accomplished by uniting a molecular microbial ecologist, a bioinformatician and a veterinarian (also a Ph.D. candidate), all of whom are new NIH Investigators, in this small-scale, health-related research project which will develop and implement novel research approaches to study fundamental aspects of infectious disease.